Actin filaments are key elements in the dynamic machinery of motile cells and in the scaffolding that maintains the shape of cells. Three cell types, each of which is specialized in such a way that separate characteristics of the actin cytoskeleton and its regulation will be studied. 1) The acrosomal reaction of Thyone sperm as a model system for investigations of the polymerization of actin. For example, how is the unpolymerized actin maintained in an insoluble state prior to induction and what role, if any, do precursor phospholipids, more specifically, phosphoinositol, play in regulation actin assembly? 2) The differentiation of hair cells of the chicken cochlea as a model for understanding how the length, number, and distribution of actin filaments is determined. First, is the length of the actin filaments regulated by capping macromolecules? This could be determined by adding exogenous gel filtered monomeric actin to detergent extracted sensory epithelia under polymerizing conditions. Those filaments that are capped should fail to elongate, while those that are uncapped will elongate. Second, when in development are new filaments added to the existing stereociliary actin bundle and where does nucleation occur? Third, how is the cuticular plate, a naturally occurring actin gel, formed? In collaboration with David DeRosier we will look at what proteins are involved and how they produce this gel. Fourth, how do actin binding proteins specifically "know" where to bind, e.g. to the stereocilia, the cuticular plate, or zonula adhaerens ring? Fifth, using in situ hybridization techniques it would be interesting to try to determine when the mRNA for actin is made in differentiating, hair cells and how different hair cells end up with the same amount of F-actin. 3) The growth and spread of the intracytoplasmic bacterial parasite, Listeria, as a model for identifying substances that nucleate the polymerization of actin and control the length of actin filaments. These studies are also of importance medically as we are studying the cell biology of entry, reproduction, and spread in host macrophages. Listeria is a model for other even more virulent bacterial such as Shigella. First, I will examine actin nucleation from the surface of Listeria by adding exogenous gel filtered actin to detergent extracted infected macrophages to see if Listeria will induce actin assembly from its surface. Second, in collaboration with Dan Portnoy, we will isolate mutant strains of Listeria that do not spread from cell to cell because they lack associated actin filaments. From these mutants I hope to identify the gene and gene products in Listeria that are needed for actin nucleation.